Modified polysaccharides exhibiting altered biological recognition

ABSTRACT

The invention relates to polysaccharides, polysaccharide conjugates and complexes with altered biological properties, and methods to produce these polysaccharides, polysaccharide conjugates and complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Ser. No. 09/555,994, filedAug. 1, 2000 now U.S. Pat. No. 6,479,468, which is a 371 ofPCT/US98/26132, filed Dec. 9, 1998, which claims benefit of priority toProvisional application Serial No. 60/069,095, filed Dec. 11, 1997 andProvisional application Serial No. 60/069,079 filed Dec. 9, 1997, whichapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Unmodified polysaccharides can have undesirable biological properties,such as rapid clearance from circulation, rapid degradation, and/orallergenicity. Two polysaccharides that are commonly employed inpharmaceutical compositions and therapeutic methods include starch anddextran.

Starch is a naturally occurring, highly biocompatible polymer. Whenstarch is introduced into the bloodstream, it is rapidly digested byamylase. The fragments of the digested product are rapidly cleared fromthe vascular compartment through glomerular filtration and/ormetabolism. For this reason hydroxyethyl starch (rather than starch) hasbeen used as a long lasting plasma volume expander for several clinicalindications. The hydroxyethylation of the starch molecule serves to slowthe rate of digestion/excretion of the polymer.

Hydroxyethylation of starch using ethylene oxide or 2-chloroethanol hasbeen common practice for production of colloidal plasma volumeexpanders. These processes have numerous disadvantages includingemploying highly toxic ethylene oxide, difficulty in controlling theextent of hydroxyethylation, inability to select among starch hydroxylgroups, toxic by products, and high cost. For example, hydroxyethylationwith ethylene oxide occurs at any hydroxylic site, including sites thathave already been hydroxyethylated, and with solvent, residual water,and impurities or side products in the reaction mixture. Lack ofselectivity among sites on the starch molecule requires extensivehydroxyethylation of the starch, although modification of certainspecific sites offers a greater degree of protection from enzymaticdegradation.

Dextran has been used for a variety of pharmaceutical and therapeuticpreparations over the past 40-50 years. The wide use of dextrans hasincluded purified native dextrans for plasma replacement/volumeexpansion, dextran-active conjugates, iron-dextran iron supplements, anddextran coated particles for MRI contrast agents. For the most partdextran in a highly purified form is well tolerated by most of thepatient population. However severe anaphylactoid responses are known tooccur, and are in some cases severe enough to result in death.

In patients undergoing hemodialysis, iron deficiency is a commonproblem. Oral iron is frequently tried first, but because of poorpatient compliance and discouraging side effects, it often fails tocorrect iron deficiency. Thus, clinicians often turn to parenteral iron.However, parenteral iron products are not without drawbacks.

Until quite recently, parenteral iron meant an iron-dextran product.Yet, iron-dextran products often cause life-threatening anaphylacticreactions. Alternatives to iron-dextran include complexes of sodiumferric gluconate sucrose and iron sucrose. While these alternativecomplexes often solve the problem of anaphylactic reactions, suchcomplexes have their own undesirable side effects.

Ferric gluconate causes transient hypotension and flushing in 1% ofexposed patients and can reoccur on re-exposure. In addition, aninteraction with ACE inhibitors leading to erythema, nausea, vomiting,cramps, and/or hypotension has been reported. Finally, rapid release ofiron from the ferrous gluconate complex can lead to circulating freeiron, with resultant oxidative stress or other possible ill effects,especially if too much iron is given at once.

In contrast, iron sucrose appears to have a reduced side-effect profile.However, as with iron gluconate, too rapid administration of too muchiron sucrose, especially in the patient with a low iron-bindingcapacity, might lead to iron oversaturation, with circulating free ironand the potential for adverse effects. Thus, like iron gluconate, ironsucrose is best given in several small doses and not in a singletotal-dose infusion.

These undesirable properties of polysaccharides employed inpharmaceutical compositions and therapeutic methods indicates the needfor modified polysaccharides, such as modified starches and modifieddextrans, that have more desirable biological properties than the nativeor unmodified polysaccharides.

SUMMARY OF THE INVENTION

The present invention relates to modified polysaccharides that have moredesirable biological properties than the native or unmodifiedpolysaccharides, pharmaceutical compositions including these modifiedpolysaccharides, methods employing these modified polysaccharides, andmethods of reducing the undesirable biological properties of thesemodified polysaccharides. Preferably, the modified polysaccharide is anoxidized and reduced polysaccharide. Preferably the polysaccharide isreduced with periodate. In a pharmaceutical composition, the oxidizedand reduced polysaccharide can be formulated in a pharmaceuticallyacceptable vehicle.

Preferred polysaccharides for modification according to the presentinvention include starch and/or dextran. An oxidized and reduced starchpreferably exhibits a longer vascular half-life than unmodified starch,slower degradation by amylase than unmodified starch, and/or slowerclearance from an animal than unmodified starch. An oxidized and reducedsoluble dextran preferably exhibits reduced allergenicity compared todextran. Preferably, the greater the extent of oxidation of thepolysaccharide, the polysaccharide exhibits a longer the vascularhalf-life, slower degradation, slower clearance, and/or lessallergenicity.

The modified polysaccharide can be a component of or be employed to forma conjugate, such as a conjugate with a chelator. A preferred chelatoris deferoxamine (DFO). The modified polysaccharide can also be used tomake pharmaceutical compositions by complexing it with micro-crystallineiron for parenteral administration.

A method of the invention includes increasing the vascular half life ofstarch by oxidizing and reducing the starch, and administering theoxidized and reduced starch into the circulation of a mammal. In anotherembodiment, the method of the invention includes decreasing theallergenicity of dextran by oxidizing and reducing the dextran, andadministering the oxidized and reduced dextran into the circulation of amammal. The dextran or starch administered can include a conjugate or acomplex of the polysaccharide with iron.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a plot of the change in the Weight Average MolecularWeight (WAMW) as a function of time for modified starches upon treatmentwith amylase.

FIG. 2 shows a plot of the average blood levels of modified starch as afunction of time.

FIG. 3 provides a plot of the change in the WAMW as a function of timefor each of the oxidized and reduced starch-DFO conjugates upontreatment with amylase.

FIG. 4 shows a plot of the average blood levels of oxidized and reducedstarch-DFO conjugates as a function of time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to modified polysaccharides that have moredesirable biological properties than the native or unmodifiedpolysaccharides, pharmaceutical compositions including these modifiedpolysaccharides, methods employing these modified polysaccharides, andmethods of reducing the undesirable biological properties of thesemodified polysaccharides.

Unmodified polysaccharides can have undesirable biological properties,such as rapid clearance from circulation, rapid degradation, and/orallergenicity. Unmodified starch, for example, is rapidly degraded andcleared from a mammal's circulation. Unmodified dextran is strongly, andperhaps fatally, allergenic in a proportion of mammals. Desirableproperties for polysaccharides include slower clearance from thecirculation, slower enzymatic digestion, and/or decreased allergenicity.Starch modified according to the present invention exhibits slowerdegradation by amylase and increased vascular half life. Dextranmodified according to the present invention exhibits decreased andacceptable allergenicity.

Modified Polysaccharides

Polysaccharides suitable for use in the present invention includedextrans and hyaluronic acid, starch and starch derivatives, and thelike. Polysaccharide starting materials such as dextrans and starchesare commercially available as water-soluble preparations or assolutions. See Remington's Pharmaceutical Sciences, A. Osol., ed., MackPublishing (16th ed. 1980) at pages 759-761. Polysaccharides of theinvention include those described in U.S. Pat. Nos. 4,863,964,5,217,998, and 5,268,165, the disclosures of which are incorporatedherein by reference.

The present invention includes a polysaccharide that has been modifiedby oxidation followed by reduction, includes a pharmaceuticalcomposition of the modified polysaccharide, includes a method formodifying a polysaccharide by oxidation and reduction, and includes amethod of controlling biological properties of a polysaccharide throughsuch modification. Oxidation followed by reduction can slow digestion,reduce allergenicity, and provide desirable alterations of otherbiological properties of the resulting biocompatible polysaccharide. Thedegree or extent to which hydroxyl groups on the polysaccharide aremodified can be easily controlled, which provides a method to select thedegree of modification of the biological property. For example, thereaction of periodate with polysaccharides is rapid and stoichiometric.The degree of oxidation of the polysaccharide molecule is homogeneousand easily and precisely controlled.

The resulting oxidized and reduced polysaccharide can be employed inmethods and compositions that currently employ native polysaccharides orconventionally modified polysaccharides. Such methods and compositionsinclude a colloidal plasma volume expander, hemodilution, a primingsolution for a heart/lung bypass machine, an organ preservationsolution, a cryoprotectant solution, and the like. Furthermore, thepresent method and modified polysaccharide provide control of the degreeof oxidation of the polysaccharide, so that the modification of thepolysaccharide can be selected to provide beneficial biologicalproperties for each of these different uses. For example, as few asabout 10% or as many as 100% of all, or of a class of, hydroxyl groupson a polysaccharide can be oxidized and reduced to alter its biologicalproperties. Preferably the hydroxyl groups are vicinal hydroxyl groups,and oxidation is accomplished employing periodate. Preferably, about 30%to about 60% of all, or of a class of, hydroxyl groups on apolysaccharide can be oxidized and reduced to slow the rate at which themodified polysaccharide is degraded, digested, or cleared from an animalcompared to the unmodified polysaccharide. Preferably, about 20% toabout 80% of all, or of a class of, hydroxyl groups on a polysaccharidecan be oxidized and reduced to reduce the allergenicity of the modifiedpolysaccharide compared to the unmodified polysaccharide.

The polysaccharide to be modified can be a component of apolysaccharide-drug conjugate, or the modified polysaccharide can beemployed to form a polysaccharide-drug conjugate or apolysaccharide-iron complex. The effects of modification according tothe invention on a polysaccharide-drug conjugate or polysaccharide-ironcomplex can be the same, e.g. slower degradation and/or reducedallergenicity, as modification of the polysaccharide itself. However,modification of a polysaccharide-drug conjugate or polysaccharide-ironcomplex, or forming a polysaccharide-drug conjugate orpolysaccharide-iron complex from a polysaccharide modified according tothe invention can have additional beneficial effects as well.

For example, the vascular half life or plasma residence time of apolysaccharide-drug conjugate or polysaccharide-iron complex may becontrolled by selection of appropriate degree of polysaccharideoxidation. When degradation of the polysaccharide or of a polysaccharideconjugate or complex controls release of the drug or iron, thisinvention provides a method for selecting controlled pharmacokinetics orsustained release of the drug. In this case, the degree ofpolysaccharide oxidation is selected for desired vascular half-life orrelease rate providing optimum effectiveness for each differentpolysaccharide-drug conjugate or polysaccharide-iron complex for eachspecific clinical indication.

Circumstances in which control of the rate of degradation of apolysaccharide conjugate can alter the effect of the conjugate or thedrug or iron released include: 1) The drug-polysaccharide conjugate orpolysaccharide-iron complex is not pharmacologically active, but aftercleavage of the backbone the fragments of the drug-polysaccharideconjugate or polysaccharide-iron complex become pharmacologicallyactive. 2) The drug-polysaccharide conjugate or polysaccharide-ironcomplex is active, and upon cleavage the fragments (active or not) aremore rapidly excreted. 3) Both the drug-polysaccharide conjugate orpolysaccharide-iron complex and their fragments are pharmacologicallyactive but increased residence time, localization orcompartmentalization of the polysaccharide-drug conjugate orpolysaccharide-iron complex and their fragments result frommodification. 4) The pharmacological profile of the active drugconjugated to the polymer changes upon cleavage of the polysaccharidebackbone. 5) Encapsulation of a drug or micro-crystalline iron within apolymeric shell of polysaccharide results in control of release of thedrug or iron from the capsule, and degradation of the shell is alteredby modification of the polysaccharide. Other polysaccharides andpolysaccharide conjugates and complexes that can benefit frommodification according to the present invention include: conjugates inwhich the active component is a nutritional compound, a pharmaceutical,an enzyme, a contrast agent, an herbicide, an insecticide, or the like;polysaccharides employed in compositions in which control of the rate ofdegradation is desired, such as with biodegradable polymers, coatings,or timed release granules.

Modified Starch

The present invention includes starch that has been modified byoxidation followed by reduction, includes a pharmaceutical compositionof the modified starch, includes a method for modifying starch byoxidation and reduction, and includes a method of controlling biologicalproperties of starch through such modification. Oxidation followed by areduction step slows digestion of the resulting biocompatible starch,and can also slow the rate of clearance from an animal. The degree orextent to which hydroxyl groups on the starch are modified can be easilycontrolled. Control and selection of the degree of oxidation determinesthe rate of enzymatic digestion (and resulting persistence orclearance-excretion) of the modified starch. Thus, the present inventionprovides a method to determine and select the vascular half life of amodified starch or of a conjugate of modified starch.

The reaction of periodate with starch is rapid (reaction goes tocompletion in a matter of minutes), specific for cleaving the bondbetween vicinal hydroxyl groups, and stoichiometric. Therefore, thedegree of oxidation of the starch molecule is homogeneous and easilycontrolled. Preferably the hydroxyl groups oxidized are vicinal hydroxylgroups, and oxidation is accomplished employing periodate.

It has been demonstrated that the oxidation/reduction of starch limitsthe rate at which it is digested by amylase. Higher degrees of oxidationof the starch molecule result in slower rates of digestion. Similarly,it has been demonstrated that oxidation/reduction of starch slows therate at which it is cleared from an animal. Higher degrees of oxidationof the starch molecule result in slower rates of clearance. By selectionof the appropriate degree of oxidation, a desired rate of digestion orclearance can be obtained. For example, as few as about 20% or as manyas 90% of all, or of a class of, hydroxyl groups on a starch can beoxidized and reduced to alter biological properties, such as its rate ofdigestion or clearance from an animal. Preferably, about 30% to about60%, of all, or of a class of, hydroxyl groups on a starch can beoxidized and reduced to slow the rate at which the modified starch isdegraded, digested, or cleared from an animal compared to the unmodifiedstarch. Under certain conditions, starch in which about 30% of all, orof a class of, hydroxyl groups on a starch are oxidized and reduced isdigested by amylase at a rate nearly as fast as unmodified starch. Underthese certain conditions, starch in which about 60% of all, or of aclass of, hydroxyl groups on a starch are oxidized and reduced is onlyvery slowly digested by amylase, and the starch can be nearly inert tosuch digestion.

The resulting oxidized and reduced starch can be employed in methods andcompositions that currently employ conventional starches, such ashydroxyethyl starch. Such methods and compositions include a colloidalplasma volume expander, for hemodilution, a priming solution for aheart/lung bypass machine, a donor organ preservation solution, and thelike. Furthermore, the present method and modified starch providecontrol of the degree of oxidation of the starch, so that themodification of the starch can be selected to provide the optimumvascular half life for each of these different uses. The starch to bemodified can be a component of a starch-drug conjugate or a starch-ironcomplex, or the modified starch can be employed to form a starch-drugconjugate or starch-iron complex. The effects of modification accordingto the invention on a starch-drug conjugate can be the same, e.g. slowerdegradation and/or reduced allergenicity, as modification of thepolysaccharide itself. However, modification of a starch-drug conjugateor starch-iron complex, or forming a starch-drug conjugate orstarch-iron complex from a starch modified according to the inventioncan have additional beneficial effects as well.

For example, the rate of excretion (or release) of a starch polymer orcomplex (or starch polymer conjugate or complex) can be controlled andselected for a particular use. Modified starch can be produced withvascular persistence selected, for example, for use as a conjugate in amedical contrast agent with a vascular half life of about 2 to about 4hours; for metal poisoning therapy, which requires the drug to remain inthe body for about 4 to about 24 hours; for plasma volume expansion,which requires a modified starch lasting about 12 to about 48 hours; orfor iron overload, which requires the modified starch to stay in thecirculation for about 2 to about 5 days. A starch that is more thanabout 30% oxidized and reduced, preferably about 30% to about 45% issuitable for the applications requiring a shorter half life. A starchthat has approaching about 60% of its hydroxyl groups oxidized andreduced, preferably about 45% to about 60% is suitable for theapplications requiring a longer half life. By way of non-limitingexample only, such highly oxidized and reduced starch may be utilized ina starch-iron complex in which slow biodegradation of the complex isdesired to avoid rapid and excessive release of iron.

The vascular half life or plasma residence time of a starch-drugconjugate or starch-iron complex may be controlled by selection ofappropriate degree of starch oxidation. When degradation of the starchof a starch conjugate or complex controls release of the drug or iron,this invention provides a method for selecting controlled or sustainedrelease of the drug or iron. In this case, the degree of starchoxidation is selected for desired vascular half-life (or release rate)providing optimum effectiveness for each different starch-drug conjugateor starch-iron complex for each specific clinical indication.

Circumstances in which control of the rate of degradation of a starchconjugate can alter the effect of the conjugate or the drug releasedinclude: 1) The drug-starch conjugate or starch-iron complex is notpharmacologically active, but after cleavage of the backbone thefragments of the drug-starch conjugate become pharmacologically active.2) The drug-starch conjugate or starch-iron complex is active, and uponcleavage the fragments (active or not) are rapidly excreted. 3) Both thedrug-starch conjugate or starch-iron complex and their fragments arepharmacologically active but increased residence time and localizationor compartmentalization of the starch-drug conjugate or starch-ironcomplex and their fragments result from modification. 4) Thepharmacological profile of the active drug conjugated to the polymerchanges upon cleavage of the starch backbone. 5) Encapsulation of drugor micro-crystalline iron within a polymeric shell of starch results incontrol of release of drug from the capsule, and degradation of theshell is altered by modification of the starch.

Modified Dextran

Chemical modification of dextran can reduce, minimize, or eliminate anallergenic reaction to native dextran, to dextran derived conjugates, orto dextran containing formulations, which are experienced in some humans(and other warm blooded species). The anaphylactic response can besevere enough to cause death, as noted in the Physicians Desk Referenceproduct information for “INFeD” (Page 2478, Edition 51, 1997).

Dextran has been used for many years for various medical therapies andcompositions, including plasma volume expansion, hemodilution andparenteral iron supplementation post hemodialysis. Dextran is oftenpreferred to other colloids used for plasma volume expansion due in partto the ability of the manufacturer to control properties such asviscosity, average molecular weight and molecular weight range, rate ofexcretion/elimination, and degree of branching, and the like. Thepresent invention offers yet another property of dextran that can bemodified and controlled to increase the usefulness of dextran, dextranformulations, and dextran conjugates and complexes.

Oxidation of the dextran molecule to produce dialdehyde-dextran,followed by a reduction step produces a modified dextran, an oxidizedand reduced dextran, which diminishes or eliminates an allergic responseto dextran. For example, as few as about 10% or as many as 100% of all,or of a class of, hydroxyl groups on a dextran can be oxidized andreduced to alter its biological properties. Preferably the hydroxylgroups are vicinal hydroxyl groups, and oxidation is accomplishedemploying periodate. Preferably, about 20% to about 80% of all, or of aclass of, hydroxyl groups on a dextran can be oxidized and reduced toreduce the allergenicity of the modified dextran compared to the dextranbefore oxidation and reduction.

The present invention can be applied to native dextran, dextran that hasalready been modified by known methods or for known purposes (such asthose described above), or to the dextran component of a conjugate. Forexample, an already formulated dextran product (such as Iron(III)Dextranfor iron supplement, or Iron-Dextran for MRI enhancement) can bemodified by oxidation and reduction to reduce or eliminate an allergenicresponse.

Although oxidation is the preferred method for modification of dextranto eliminate or reduce the allergenic response, other methods ofchemical modification are also effective. For example, suitablemodifications may include hydroxyethylation, alkylation, reduction,esterfication, and the like.

Method for Modifying Polysaccharide by Oxidation and Reduction

Modification of a polysaccharide, such as starch or dextran, can becarried out by dissolving the polysaccharide, such as starch or dextran,in an aqueous medium at a concentration of approximately 100 g/L. Whilestirring, a suitable oxidizing agent, preferably periodate, preferably asolution of NaIO₄, NaIO₄ solid, or HIO₄, is added to oxidize thepolysaccharide, such as starch or dextran. The amount of oxidizingagent, such as NaIO₄, used will control the amount of oxidation ormodification of the polysaccharide, such as starch or dextran. Thereaction mixture is then purified by conventional methods to removesalts from the oxidation reaction and to recover the polysaccharide,such as starch or dextran, with dialdehyde and other aldehyde moietiesformed by oxidation. Next, the polysaccharide, such as starch ordextran, with dialdehyde and other aldehyde moieties formed by oxidationare reduced. A preferred reducing agent is NaBH₄. The dialdehydefunctional groups are thereby converted to dialcohol groups, andaldehydes are reduced as well. Finally, the reaction mixture is againpurified by conventional methods to remove salts from the reactionmixture.

Additional suitable methods for oxidizing and reducing polysaccharides,such as starch and dextran, are described in U.S. patent applicationSer. No. 08/911,991, to be issued as U.S. Pat. No. 5,847,110 on Dec. 8,1998, the disclosure of which is incorporated herein by reference.

The oxidized and reduced polysaccharide, such as oxidized and reducedstarch or oxidized and reduced dextran, is then ready for formulation.Alternatively, prior to reduction of the oxidized polysaccharide, suchas oxidized starch or oxidized dextran, the aldehyde functional groupson the oxidized polysaccharide, such as oxidized starch or oxidizeddextran, can be conjugated to a drug, a chelator, or another activemoiety to produce a conjugate.

The present method of oxidation and reduction has several advantagescompared to other, previously available methods for modification ofpolysaccharides to alter their biological properties. Compared to, forexample, hydroxyethylation, the degree of oxidation is much easier tocontrol since the reaction with oxidizer is stoichiometric. Oxidation ismore specific, typically being limited to only one site on a glucose, orother saccharide, subunit. In addition, reaction by-products of theoxidation reaction have low toxicity and are easily removed. Oxidationis cost effective since an oxidant such as periodic acid may beregenerated by an electrolytic process.

Conjugates Employing Oxidized and Reduced Polysaccharide

Covalently binding a chelator, or other molecule, to a polysaccharidehas been discovered to be advantageous for several reasons. For example,binding to the polysaccharide can alter the distribution of thechelator, or other molecule, in the patient. For example, although notlimiting to the present invention, it is believed that the conjugate ofa chelator and polysaccharide is retained in the circulation to agreater degree than the chelator alone. Additional advantageous featuresof a conjugate can include altered biodistribution, diminished toxicity,increased stability of the chelator, or other molecule, in solution,formulations and plasma, and greater efficacy of the chelator, or othermolecule.

The polysaccharide is preferably an oxidized and reduced polysaccharide.Polysaccharides suitable for oxidation and reduction include dextransand hyaluronic acid, starch and starch derivatives, and the like.Polysaccharide starting materials such as dextrans and starches arecommercially available as water-soluble preparations or as solutions.See Remington's Pharmaceutical Sciences, A. Osol., ed., Mack Publishing(16th ed. 1980) at pages 759-761. Polysaccharides of the inventioninclude those described in U.S. Pat. Nos. 4,863,964, 5,217,998, and5,268,165, the disclosures of which are incorporated herein byreference.

The oxidized and reduced polysaccharide is sufficiently stable to carrythe chelator, or other molecule, in the patient for a sufficient timethat it is effective for the desired biological or therapeutic purpose.In addition, the polysaccharide is sufficiently well-tolerated andnon-toxic (e.g. nonallergenic) that the patient has no unacceptableadverse reactions to the treatment.

Preparing a Conjugate

There are several ways in which a chelator, or other molecule, can becovalently bonded to the polysaccharide or oxidized polysaccharide toform a conjugate. The chelator, or other molecule, is bound to thepolysaccharide or oxidized polysaccharide in a manner such that itsdesired properties, as measured in vitro, remain substantial, andpreferably on the order of the non-conjugated chelator, or othermolecule. One preferred way to form conjugates of a chelator, or othermolecule, with a polysaccharide or oxidized polysaccharide is to bind anamino group, such as a terminal amino group of deferoxamine, to thepolysaccharide or oxidized polysaccharide. Such an amino group can forma covalent bond with a carboxyl group on a polysaccharide to form anamide linkage.

Preferably, an amino group of the chelator, or other molecule, will forma covalent bond with an aldehyde moiety. In an initial reaction, theamine on the chelator, or other molecule, reacts with the aldehyde toform a Schiff base, and the Schiff base is reduced in a second reactionto yield more stable covalent linkage. Aldehydic groups can beintroduced into the polysaccharide by known techniques, e.g. by theoxidation of carbohydrates or other diols to dialdehydes with sodiummetaperiodate. See, for example, M. B. Wilson, et al. inImmunofluorescence and Related Staining Techniques, W. Knapp et al.,eds., Elsevier/North Holland Biomedical Press (1978) at page 215,Flemming et al., Acta Biol. Med. Ger., 30, 177 (1973); and, S.-C. Tam etal., in P.N.A.S. USA, 73, 2128 (1976). In some applications, theterminal amino group on a chelator, or other molecule, can also bebonded to an amino group on the polymer directly, by the use of adialdehyde linking agent such as glutaraldehyde, followed by reduction,e.g., with sodium borohydride.

More preferred chelator conjugates are prepared by covalently bondingdeferoxamine to a pharmaceutically-acceptable polysaccharide or oxidizedpolysaccharide. Methods for the preparation of deferoxamine(N-[5-[3[(5-aminopentyl) hydroxycarbamoyl]propionamido]pentyl]-3-[[5-(N-hydroxyacetamido) pentyl]carbamoyl]propionohydroxamic acid) and its pharmaceutically-acceptablesalts have been disclosed, e.g., by Prelog et al., in Helv. Chim. Acta.,45, 631 (1962); Bickel et al., Helv. Chim. Acta, 46 1385 (1963); inGerman Pat. Spec. 1,186,076 and in U.S. Pat. Nos. 4,419,365, 4,987,253,and 5,493,053, the disclosures of which are incorporated by referenceherein. Such salts include the acid addition salts of methane sulfonicacid, phosphoric acid, acetic acid, lactic acid, tartaric acid, citricacid, and the like. Other suitable chelators include2,3-dihydroxybenzoic acid, DTPA, rhodotorulic acid, cholylhydroxamicacid, ethylene diamine-N,N′-bis(2-hydroxyphenylacetic acid),isoniazid-pyridoxal hydrozone, 1,2-dimethyl-3-hydroxypyrid-4-one andnitrilotriacetate. These chelators can be used alone or in combination.

Methods for preparing chelator conjugates include the methods describedin U.S. Pat. Nos. 4,863,964, 5,217,998, and 5,268,165, and in U.S.patent application Ser. No. 08/911,991, to be issued as U.S. Pat. No.5,847,110 on Dec. 8, 1998, the disclosures of which are incorporatedherein by reference.

The mole ratios of chelator or other molecule to polysaccharideattainable by reactions with carboxyl or carbonyl groups can varywidely, depending on factors such as the number of reactive groups onthe polymer, steric hindrance, rate and extent of Schiff base or amideformation, and the like. More than one molecule of chelator or othermolecule can be attached to each molecule of polysaccharide. As anexample, about 0.7 g of deferoxamine can be bonded to about 2.5 g ofreacted Dextran 40, via reaction of the deferoxamine with aldehydegroups introduced into the dextran, followed by reduction.

Preparing a Complex

Processes for forming iron-dextran complexes are well-known in the art.Monte el al. describe such a process in U.S. Pat. No. 5,756,715, whichis fully incorporated herein by reference. In sum, that processcomprises the steps of combining an aqueous solution of dextran with anaqueous solution of a iron (III) salt, combining the mixture withalkali, heating and stirring the mixture until formation of aniron-dextran complex is substantially complete, acidifying the mixture,precipitating the iron-dextran complex by adding the mixture to a watermiscible solvent, and isolating the crystalline iron-dextran complex.This basic methodology can also be employed to form iron-starchcomplexes.

Administering the Oxidized and Reduced Polysaccharide

The oxidized and reduced polysaccharide conjugate or complex can bedelivered by a variety of routes effective to gain circulating and locallevels sufficient to provide the desired biological or therapeuticeffect. Typical routes of administration would be parenteral, such asintravenous or subcutaneous. The oxidized and reduced polysaccharide ispreferably administered as a solution or suspension in an aqueoussolvent that is compatible with administration to patients such asanimals, mammals or humans. A preferred pharmaceutical composition isnon-pyrogenic. Preferably oxidized and reduced polysaccharide isadministered, as solutions, parenterally, such as by intramuscular,intraperitoneal, subcutaneous, intraocular, or intravenous injection orinfusion, or via buccal, oral, pulmonary, rectal or vaginal routes. Theappropriate dose will be adjusted in accord with appropriate clinicalfactors in the treating physician's judgment including: the disorder tobe treated; the patient or patient's age, size and weight; the mode ofadministration; properties of the particular oxidized and reducedpolysaccharide, and the like.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example 1—Modification of Native Starch

The native starch employed was unmodified degraded waxy maize starch (MW126,000), which was obtained from Laevosan (Linz, Austria) and is Lot43572, PN 1021B. All water used for preparation of these modifiedpolysaccharides was depyrogenated water, and all other vessels/utensilswere depyrogenated prior to use. When possible, reactions and work-upswere performed in the laminar flow hood to preserve purity of thesesolutions, for intravenous and other parenteral administration.Reactions were performed at ambient room temperature and cooling wasapplied in some cases to prevent warming of the reaction mixture.

Experiment 1: Preparation of Modified Starch With 175 mM Periodate

In a clean, glass vessel 30 g of starch powder was dissolved in 300 mLof water. To this was slowly added 11.23 g of NaIO₄ (175 mM), and thismixture was stirred for 105 minutes. The resulting mixture wasdiafiltered (membrane: Pellicon 2 mini, 5K MWCO, 0.1 m²) against wateruntil the conductivity of the filtrate was 25 μS. The oxystarchconcentration was then adjusted to 100 g/L. While stirring NaBH₄ (2.38g) was added to the oxystarch solution (239 mL) to obtain a NaBH₄concentration of 263 mM. The reaction was stirred for 2 hours. Theresulting solution was diafiltered against water until the filtrateconductivity reaches 58 μS. The pH of the solution was then adjusted to6.3 with HCl, and polymer concentration was adjusted to 102 g/L.Finally, the chloride concentration was adjusted to 154 mM. The solutionwas then sterile filtered into glass vials, stoppered and stored at 4°C. This yielded approximately 192 mL of modified starch product in whichapproximately 28% of the glucose sub-units were modified.

Experiment 2: Preparation of Modified Starch With 222 mM Periodate

In a clean, glass vessel 30 g of starch powder was dissolved in 300 mLof water. To this was slowly added 14.25 g of NaIO₄ (222 mM), and themixture was stirred for 60 minutes. The resulting mixture wasdiafiltered (membrane: Pellicon 2, 5K MWCO, 0.5 m²) against water untilthe conductivity of the filtrate was 27 μS. The oxystarch concentrationwas then adjusted to 100 g/L. While stirring NaBH₄ (2.27 g) was added tothe oxystarch solution (180 mL) to obtain a NaBH₄ concentration of 333mM. The reaction was stirred for 190 minutes. The resulting solution wasdiafiltered against water until the filtrate conductivity reaches 35 μS.The pH of the solution was then adjusted to 6.5 with HCl, and polymerconcentration was adjusted to 100 g/L. Finally, the chlorideconcentration was adjusted to 154 mM. The solution was then sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 133 mL of modified starch product in which approximately36% of the glucose sub-units were modified.

Experiment 3: Preparation of Modified Starch With 278 mM Periodate

In a clean, glass vessel 30 g of starch powder was dissolved in 300 mLof water. To this was slowly added 17.84 g of NaIO₄ (278 mM), and themixture was stirred for 90 minutes. The resulting mixture wasdiafiltered (membrane: Pellicon 2, 5K MWCO, 0.5 m²) against water untilthe conductivity of the filtrate was 13 μS. The oxystarch concentrationwas then adjusted to 100 g/L. While stirring NaBH₄ (3.09 g) was added tothe oxystarch solution (196 mL) to obtain a NaBH₄ concentration of 417mM. The reaction was stirred for 185 minutes. The resulting solution wasdiafiltered against water until the filtrate conductivity reaches 45 μS.The pH of the solution was then adjusted to 5.5 with HCl, and polymerconcentration was adjusted to 100 g/L. Finally, the chlorideconcentration was adjusted to 154 mM. The solution was then sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 151 mL of modified starch product in which approximately45% of the glucose sub-units were modified.

Experiment 4: Preparation of Modified Starch With 334 mM Periodate

In a clean, glass vessel 30 g of starch powder was dissolved in 300 mLof water. To this was slowly added 21.43 g of NaIO₄ (334 mM), and themixture was stirred for 105 minutes. The resulting mixture wasdiafiltered (membrane: Pellicon 2, 5K MWCO, 0.5 m²) against water untilthe conductivity of the filtrate was 43 μS. The oxystarch concentrationwas then adjusted to 100 g/L. While stirring NaBH₄ (3.66 g) was added tothe oxystarch solution (193 mL) to obtain a NaBH₄ concentration of 501mM. The reaction was stirred for 185 minutes. The resulting solution wasdiafiltered against water until the filtrate conductivity reached 35 μS.The pH of the solution was then adjusted to 4.4 with HCl, and polymerconcentration was adjusted to 100 g/L. Finally, the chlorideconcentration was adjusted to 154 mM. The solution was then sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 127 mL of modified starch product in which approximately54% of the glucose sub-units were modified.

Example 2—Oxidation and Reduction of Starch Slows Amylase Digestion

Modified starch products prepared above in Experiments 2, 3 and 4 ofExample 1 were examined for rate of digestion by α-amylase using gelpermeation chromatography with refractive index and laser lightscattering detection.

A solution of α-amylase (Sigma A-6255, Lot 33H8075, from porcinepancreas, Type 1-A DFP treated, 30 mg protein/mL, 790 units/mg protein)was prepared by diluting the stock solution 1:50 with 0.9% aqueous NaClsolution. The digestion samples were prepared in a glass test tube inthe following manner: Into the tube were placed 0.5 mL of 0.9% aqueousNaCl, 0.1 mL of 50 mM CaCl₂, 0.1 mL HEPES (pH 7), and 10 μL of α-amylasesolution (prepared above) and mixed well. Next was added 10.0 mL ofmodified (oxidized and reduced) starch (100 g/L) to the test tube, whichwas vortexed and allowed to stand at room temperature. At given timepoints 200 μL of amylase/starch solution was sampled, diluted with 0.88mL of gel permeation chromatography (GPC) eluent and mixed well. Thiswas then injected on a GPC column for measurement of the samples'molecular weight distribution.

FIG. 1 illustrates a plot of the change in the Weight Average MolecularWeight (WAMW) as a function of time for each of the modified starchproducts. These data are given in Table 1.

TABLE 1 Change in WAMW Over Time As a Percentage of Initial WAMW 222 mMNaIO₄ 278 mM NaIO₄ 334 mM NaIO₄ Time (hr) (36% Modified) (45% Modified)(54% Modified) 0.00 100% 100% 100% 0.08 94% 93% 99% 3.00 69% 84% 97%7.00 57% 78% 95% 24.00 38% 59% 89%

In each experiment, oxidation and reduction of starch slowed itsdegradation by amylase.

Example 3—Oxidized and Reduced Starch is Cleared More Slowly in Animals

In-vivo blood clearance in rats of several modified (oxidized andreduced) starches was examined. Modified starch products prepared abovein Experiments 2 through 4 were administered i.v. (femoral vein) toSprague-Dawley rats at a dosage of 10 mL/kg given as a bolus injection.Starch samples modified with 222 mM and 278 mM NaIO₄ were each injectedinto three animals. Two animals were injected with starch modified with334 mM NaIO₄. Samples of blood were drawn from the femoral vein at 30minutes, 1 hr, 2 hr, 4 hr and 8 hr. These samples were then assayed formodified starch concentration.

FIG. 2 shows a plot of the average blood levels of modified starch as afunction of time. These data are provided in Table 2.

TABLE 2 Average Blood Levels of Modified Starch Products (n = 3 animals)As a Percentage of Initial Dose Remaining in the Plasma 222 mM NaIO₄ 278mM NaIO₄ 334 mM NaIO₄ Time (hr) (36% Modified) (45% Modified) (54%Modified) 0.5 33.1% 47.0% 67.7% 1 20.8% 37.9% n/a 2 12.6% 28.4% 48.1% 47.4% 18.6% 39.7% 8 4.3% 12.3% 29.6%

In each experiment, oxidation and reduction of starch slowed itsclearance from the animal.

Example 4—Modification of Starch in Synthesis of a Starch-DeferoxamineConjugate

The next several experiments detail preparation of DFO-starch productsand oxidation and reduction of the starch or conjugate. Each usedunmodified degraded waxy maize starch (starch) MW 126,000 or 46,000 anddeferoxamine mesylate (DFO) as raw materials. The starch was obtainedfrom Laevosan (Linz, Austria). The DFO was obtained from Pharmacia &Upjohn (Kalamazoo, Mich.) Lot 22ADF, PN 1001B. All water used forpreparation of these batches is depyrogenated water, and all othervessels/utensils were depyrogenated prior to use. When possible,reactions and work-ups were performed in the laminar flow hood topreserve purity of these solutions. All reactions were performed at orslightly below ambient room (laboratory) temperature.

Experiment 1: Preparation of DFO-Starch Conjugate With 34 mM Chelatorand 150 mM Periodate

In a clean, glass vessel 99.8 g of starch powder (MW 46,000) wasdissolved in 1000 mL of water. Next, 32.0 g of NaIO₄ (150 mM) was addedto the mixture, and stirred for 90 minutes. The resulting mixture wasdiafiltered (membrane: Biomax 5K MWCO) against water until theconductivity of the filtrate was 103 μS. The oxystarch concentration wasthen adjusted to 245 g/L. The reaction volume was adjusted to 288 mLwith water and ethanol to provide a mixture that was 30% ethanol byvolume. DFO (22.26 g) was added while stirring. Stirring was continuedfor 1 hour, after which 5.65 mL of 8M borane pyridine complex (BPC) wasadded to the reaction mixture. The mixture was then stirred for 20hours. At the end of this reaction period, an additional 144 mL of waterwas added to the mixture followed by slow addition of 3.87 g NaBH₄ (225mM). Stirring was continued for 1 hour, after which the reaction mixturewas diafiltered against water until the filtrate conductivity was 30 μS.The pH was then adjusted to 6.0 with HCl, the chloride concentration wasadjusted to provide a final concentration of 154 mM, and the polymerconcentration was adjusted to 100 g/L. Finally, the solution was sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 515 mL of starch-DFO product in which approximately 24% ofthe glucose sub-units were modified, and had a high molecular weightchelator concentration of 34 mM at a starch-DFO concentration of 100g/l.

Experiment 2: Preparation of DFO-Starch Conjugate With 45 mM Chelatorand 550 mM Periodate

In a clean, glass vessel 100 g of starch powder (MW 46,000) wasdissolved in 970 mL of water. Next, 117.7 g of NaIO₄ (550 mM) was addedto the mixture, and stirred for 60 minutes. The resulting mixture wasdiafiltered (membrane: Pellicon 2 Biomax 5K MWCO) against water untilthe conductivity of the filtrate was 167 μS. The oxystarch concentrationwas then adjusted to 166 g/L. The reaction volume was adjusted to 540 mLwith water and ethanol to provide a mixture that was 30% ethanol byvolume. DFO (27.09 g) was added while stirring. Stirring was continuedfor 1 hour, after which 13.8 mL of 8M borane pyridine complex (BPC) wasadded to the reaction mixture. The mixture was then stirred for 18hours, after which 7 mL of BPC was added and stirring continued foranother 2 hours. At the end of this reaction period, an additional 572mL of water was added to the mixture followed by slow addition of 17.17g NaBH₄ (825 mM). Stirring was continued for 3 hours, after which thereaction mixture was diafiltered against water until the filtrateconductivity was 124 μS. The pH was then adjusted to 6.0 with HCl, thechloride concentration was adjusted to provide a final concentration of154 mM, and the polymer concentration was adjusted to 100 g/L. Finally,the solution was sterile filtered into glass vials, stoppered and storedat 4° C. This yielded approximately 515 mL of starch-DFO product inwhich approximately 89% of the glucose sub-units were modified, and hada high molecular weight chelator concentration of 45 mM at a starch-DFOconcentration of 100 g/l.

Experiment 3: Preparation of DFO-Starch Conjugate With 36 mM Chelatorand 150 mM Periodate

In a clean, glass vessel 50 g of starch powder (MW 126,000) wasdissolved in 465 mL of water. Next 16.0 g of NaIO₄ (150 mM) was added tothe mixture, and stirred for 60 minutes. The resulting mixture wasdiafiltered (membrane: Pellicon 2 mini, 5K MWCO) against water until theconductivity of the filtrate was 136 μS. The oxystarch concentration wasthen adjusted to 165 g/L. The reaction volume was adjusted to 385 mLwith water and ethanol to provide a mixture that was 30% ethanol byvolume. DFO (19.51 g) was added while stirring. Stirring was continuedfor 5 minutes, after which 4.95 mL of 8M borane pyridine complex (BPC)was added to the reaction mixture. The mixture was then stirred for 19hours. At the end of this reaction period 3.39 g NaBH₄ (225 mM) wasslowly added to the reaction vessel while stirring. Stirring wascontinued for 90 minutes, after which the reaction mixture wasdiafiltered against water until the filtrate conductivity was 97 μS. ThepH was then adjusted to 6.0 with HCl, the chloride concentration wasadjusted to provide a final concentration of 154 mM, and the polymerconcentration was adjusted to 100 g/L. Finally, the solution was sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 470 mL of starch-DFO product in which approximately 24% ofthe glucose sub-units were modified, and had a high molecular weightchelator concentration of 36 mM at a starch-DFO concentration of 100g/l.

Experiment 4: Preparation of DFO-Starch Conjugate With 47 mM Chelatorand 550 mM Periodate

In a clean, glass vessel 50 g of starch powder (MW 126,000) wasdissolved in water to a final volume of 500 mL. Next 58.8 g of NaIO₄(550 mM) was added to the mixture, and stirred for 60 minutes. Theresulting mixture was diafiltered (membrane: Pellicon 2 mini, 5K MWCO)against water until the conductivity of the filtrate was 167 μS. Theoxystarch concentration was then adjusted to 160 g/L. The reactionvolume was adjusted to 395 mL with water and ethanol to provide amixture that was 30% ethanol by volume. DFO (19.28 g) was added whilestirring. Stirring was continued for 60 minutes, after which 14.7 mL of8M borane pyridine complex (BPC) was added to the reaction mixture. Themixture was then stirred for 20 hours. At the end of this reactionperiod 12.2 g NaBH₄ (825 mM) was slowly added to the reaction vesselwhile stirring. Stirring was continued for 180 minutes, after which thereaction mixture was diafiltered against water until the filtrateconductivity was 144 μS. The pH was then adjusted to 6.0 with HCl, thechloride concentration was adjusted to provide a final concentration of154 mM, and the polymer concentration was adjusted to 100 g/L. Finally,the solution was sterile filtered into glass vials, stoppered and storedat 4° C. This yielded approximately 517 mL of starch-DFO product inwhich approximately 89% of the glucose sub-units were modified, and hasa high molecular weight chelator concentration of 47 mM at a starch-DFOconcentration of 100 g/l.

Experiment 5: Preparation of DFO-Starch Conjugate With 40 mM Chelatorand 222 mM Periodate

In a clean, glass vessel 50 g of starch powder (MW 126,000) wasdissolved in 473 mL of water. Next 23.74 g of NaIO₄ (222 mM) was addedto the mixture, and stirred for 65 minutes. The resulting mixture wasdiafiltered (membrane: Biomax Pellicon 2 mini, 5K MWCO) against wateruntil the conductivity of the filtrate was 90 μS. The oxystarchconcentration was then adjusted to 163 g/L. The reaction volume wasadjusted to 388 mL with water and ethanol to provide a mixture that was30% ethanol by volume. DFO (19.47 g) was added while stirring. Stirringwas continued for 15 minutes, after which 7.42 mL of 8M borane pyridinecomplex (BPC) was added to the reaction mixture. The mixture was thenstirred for 20 hours. At the end of this reaction period 4.99 g NaBH₄(333 mM) was slowly added to the reaction vessel while stirring.Stirring was continued for 200 minutes, after which the reaction mixturewas diafiltered against water until the filtrate conductivity was 96 μS.The pH was then adjusted to 6.0 with HCl, the chloride concentration wasadjusted to provide a final concentration of 154 mM, and the polymerconcentration was adjusted to 100 g/L. Finally, the solution was sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 529 mL of starch-DFO product in which approximately 36% ofthe glucose sub-units were modified, and had a high molecular weightchelator concentration of 40 mM at a starch-DFO concentration of 100g/l.

Experiment 6: Preparation of DFO-Starch Conjugate With 42 mM chelatorand 278 mM Periodate

In a clean, glass vessel 50 g of starch powder (MW 126,000) wasdissolved in 445 mL of water. Next 29.71 g of NaIO₄ (278 mM) was addedto the mixture, and stirred for 65 minutes. The resulting mixture wasdiafiltered (membrane: Biomax Pellicon 2 mini, 5K MWCO) against wateruntil the conductivity of the filtrate was 107 μS. The oxystarchconcentration was then adjusted to 169 g/L. The reaction volume wasadjusted to 393 mL with water and ethanol to provide a mixture that was30% ethanol by volume. DFO (19.72 g) was added while stirring. Stirringwas continued for 18 minutes, after which 10.00 mL of 8M borane pyridinecomplex (BPC) was added to the reaction mixture. The mixture was thenstirred for 20 hours. At the end of this reaction period 6.39 g NaBH₄(417 mM) was slowly added to the reaction vessel while stirring.Stirring was continued for 180 minutes, after which the reaction mixturewas diafiltered/against water until the filtrate conductivity was 91 μS.The pH was then adjusted to 6.0 with HCl, the chloride concentration wasadjusted to provide a final concentration of 154 mM, and the polymerconcentration was adjusted to 100 g/L. Finally, the solution was sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 558 mL of starch-DFO product in which approximately 45% ofthe glucose sub-units were modified, and had a high molecular weightchelator concentration of 42 mM at a starch-DFO concentration of 100g/l.

Experiment 7: Preparation of DFO-Starch Conjugate With 43 mM Chelatorand 334 mM Periodate

In a clean, glass vessel 50 g of starch powder (MW 126,000) wasdissolved in 450 mL of water. Next 35.72 g of NaIO₄ (334 mM) was addedto the mixture, and stirred for 60 minutes. The resulting mixture wasdiafiltered (membrane: Biomax Pellicon 2 mini, 5K MWCO) against wateruntil the conductivity of the filtrate was 110 μS. The oxystarchconcentration was then adjusted to 181 g/L. The reaction volume wasadjusted to 335 mL with water and ethanol to provide a mixture that was30% ethanol by volume. DFO (16.98 g) was added while stirring. Stirringwas continued for 15 minutes, after which 10.80 mL of 8M borane pyridinecomplex (BPC) was added to the reaction mixture. The mixture was thenstirred for 20 hours. At the end of this reaction period 6.54 g NaBH₄(501 mM) was slowly added to the reaction vessel while stirring.Stirring was continued for 170 minutes, after which the reaction mixturewas diafiltered against water until the filtrate conductivity was 108μS. The pH was then adjusted to 6.0 with HCl, the chloride concentrationwas adjusted to provide a final concentration of 154 mM, and the polymerconcentration was adjusted to 100 g/L. Finally, the solution was sterilefiltered into glass vials, stoppered and stored at 4° C. This yieldedapproximately 474 mL of starch-DFO product in which approximately 54% ofthe glucose sub-units were modified, and had a high molecular weightchelator concentration of 43 mM at a starch-DFO concentration of 100g/l.

Example 5—Oxidation and Reduction of Starch Slows Amylase Digestion ofDFO-Starch Conjugates

DFO-conjugates prepared above in Experiments 3 and 4 of Example 4 wereexamined for rate of digestion by α-amylase using gel permeationchromatography with refractive index and laser light scatteringdetection.

The digestion samples were prepared in a manner similar to the procedureoutline in Example 2. The digested samples were injected on the GPCcolumn for measurement of the sample molecular weight distribution.

FIG. 3 provides a plot of the change in the Weight Average MolecularWeight (WAMW) as a function of time for each of the modified starchproducts. These data are given in Table 3.

TABLE 3 Decrease in Weight Average Molecular Weight Upon Incubation withAmylase Expressed as a Percentage of Initial WAMW 150 mM NaIO₄ 550 mMNaIO₄ Time (hr) (˜24% Modified) (˜89% Modified) 0.00 100.0% 100.0% 0.0891.0% n/a 1 72.7% 95.6% 2 n/a 95.6% 3 43.7% n/a 7 n/a 95.6% 23 n/a 95.6%25 17.0% n/a 168 n/a 95.6% n/a-not available

In each experiment, an increased extent of oxidation and reduction ofstarch slowed the rate of degradation by amylase of the starch-DFOconjugate.

Example 6—Increasing the Oxidation and Reduction of a Starch-DFOConjugate Slows Clearance in Animals

In-vivo blood clearance in rats of several DFO-oxidized and reducedstarch conjugates was examined. Several DFO-oxidized and reduced starchconjugates as prepared above in Experiments 1 through 7 of Example 4were administered i.v. (femoral vein) to Sprague-Dawley rats at a dosageof 10 mL/kg given as a 30 minute infusion. In all cases, either two orthree animals were used for this experiment. Samples of blood were drawnfrom the femoral vein at various time points. These samples were thenassayed for DFO-Starch conjugate concentration.

FIG. 4 shows a plot of the average blood levels of DFO-Starch conjugateas a function of time. Experiments 1 and 2 employed starch having amolecular weight of 46,000. Experiments 3-7 employed starch having amolecular weight of 126,000. These data are provided in Table 4.

TABLE 4 Average Blood Clearance of DFO-Starch Conjugate Products As aPercentage of Plasma Concentration at 30 min. Exp. 1 Exp. 2 Exp. 3 Exp.4 Exp. 5 Exp. 6 Exp. 7 Time 24% 89% 24% 89% 36% 45% 54% (hr) Mod Mod ModMod Mod Mod Mod 0.5 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 115.3% 76.0% 33.7% 96.0% 66.4% 78.8% 90.1% 2 2.0% 49.7% 11.9% 71.9% 41.6%55.9% 75.6% 4 0.6% 36.9% 2.3% 60.5% 23.8% 37.2% 53.2% 8 n/a n/a n/a n/a13.0% 18.7% 32.8% n/a-not available

An increased extent of oxidation and reduction of the starch slowedclearance of the starch-DFO conjugate from the animal.

Example 7—Modification of Dextran

Dextran modification was carried out by a slight modification of themethod described in Example 1 for oxidizing starch. Briefly, dextran wasdissolved in an aqueous medium at a concentration of approximately 100g/L and while stirring, then was added a solution of NaIO₄ (or NaIO₄ insolid form) to oxidize the dextran. The amount of NaIO₄ used controlledthe amount of dextran oxidation. The reaction mixture was then purifiedto remove salts from the oxidation reaction. Next, the resultantdialdehyde groups (formed by the oxidation reaction) were reduced toalcohol groups using NaBH₄. Finally, the reaction mixture was againpurified to remove salts from the reaction mixture. The “modifieddextran” was then ready for formulation or further modification.

Example 8—Reduced Allergenicity of Oxidized and Reduced Dextran

Oxidized and reduced dextran was administered i.v. to Sprague-Dawleyrats at a dosage of 40 mL/kg over 30 minutes. About 100% of the vicinalhydroxyls on the dextran had been oxidized with periodate and reduced. Avery minor allergic reaction was observed as demonstrated by slightinflation of the front paw pads (but not rear paw pads). After dosing,the animal fully recovered from anesthesia.

In contrast, when an equivalent dose of native dextran was infused intoa rat a very severe allergic reaction was observed after 15 minutes,approximately half way through the dose, the paw pads (front and rear)swelled, and the dosing was halted. Soon after termination of dosing,the animal's tail and body became severely edematous, and the animaldied at this point. It is believed that the death was caused bysuffocation due to constriction of the air passageway brought on by anallergic reaction to the dextran. This strain of rat is known to beallergic to dextran.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention. All publications andpatent applications in this specification are indicative of the level ofordinary skill in the art to which this invention pertains.

Example 9—Preparation of a Modified Starch-Iron Complex

One hundred (100) grams of water-soluble rich starch was dissolved inone liter purified water. It was oxidized with 53.5 grams of sodiummetaperiodate for one hour at room temperature. Following diafiltration,the resulting aldehyde groups were reduced by adding 14.3 grams ofsodium borohydride. This yielded a modified starch product in whichapproximately 40% of the glucose sub-units were modified. The resultingoxidized and reduced starch was purified by diafiltration and adjustedto 100 g/Liter. A solution of iron chloride was prepared by dissolving3.0 grams of iron (III) chloride (hexahydrate) in 35 ml water. To thissolution was slowly added a solution of 20 ml sodium carbonatecontaining 0.59 g of anhydrous Na₂CO₃. While stirring, 70 ml of themodified starch solution was added to the buffered iron chloride-sodiumcarbonate solution. Using a buret, approximately 50 ml of 0.5 N NaOH wasadded, with vigorous stirring, to the starch-iron solution. Theresulting solution was then poured into 350 ml isopropanol with stirringto precipitate the starch iron complex. The precipitate is then filteredon a Whitman #1 filter. The filtrate is transferred to a beakercontaining 75 ml water. The precipitate is dissolved by stirring andheating to 90° C. for sixty minutes. The material is once againprecipitated in isopropyl alcohol, filtered and redissolved as above toyield a purified modified starch-iron complex.

We claim:
 1. A pharmaceutical composition comprising a complex ofpartially oxidized and reduced water-soluble polysaccharide andmicro-crystalline iron, wherein the polysaccharide is unmodified starchand wherein about 20% to 80% of vicinal hydroxyl groups of thepolysaccharide have been oxidized, and a pharmaceutically acceptablevehicle.
 2. The pharmaceutical composition of claim 1, wherein theextent of oxidation and reduction of the soluble starch is effective toprovide a longer vascular half-life than native starch.
 3. Thepharmaceutical composition of claim 1, wherein about 30% to about 60% ofvicinal hydroxyl groups of the polysaccharide have been oxidized.
 4. Thepharmaceutical composition of claim 1, wherein about 40% of vicinalhydroxyl groups of the polysaccharide have been oxidized.
 5. A method ofincreasing the vascular half life of a starch-iron complex comprisingthe steps of: a. oxidizing about 20% to about 90% of the vicinalhydroxyl groups of unmodified starch; b. reducing the oxidized starch;c. forming a starch-iron complex using the oxidized and reduced starch;and d. administering the oxidized and reduced starch-iron complex intothe circulation of a mammal.